Biochemical analyses are invaluable, routine tools in health-related fields such as immunology, pharmacology, gene therapy, and the like. In order to successfully implement therapeutic control of biological processes, it is imperative that an understanding of biological binding between various species is gained. Indeed, an understanding of biological binding between various species is important for many varied fields of science.
Many biochemical analytical methods involve immobilization of a biological binding partner of a biological molecule on a surface, exposure of the surface to a medium suspected of containing the molecule, and determination of the existence or extent of molecule coupling to the surface-immobilized binding partner.
The study of biological binding involving nucleic acid at surfaces has been hindered by the difficulty in immobilizing a single strand of nucleic acid at a surface without also immobilizing the complement of that strand. Where a single strand of nucleic acid is immobilized at a surface with its complement, it is not available for interaction by itself.
Electron transfer through model enzymes has been studied, and several theoretical models predict rates of transfer through these enzymes (Chidsey, C.E.D., "Free Energy and Temperature Dependence of Electron Transfer at the Metalelectrolyte Interface," Science 251 (1991), pp. 919-922). Comparison of predicted electron transfer rates with the time required for electrons to travel a finite distance within a protein has led to the conclusion that electrons traverse a pathway of chemical bonds such as covalent or hydrogen bonds (J. N. Onuchic, D. N. Beratan, J. R. Winkler, and H. B. Gray, Ann. Rev. Biophys. Biomol. Struct., 21 349 (1992); D. N. Baratan, J. N. Onuchic, J. R. Winkler and H. B. Gray, Science, 258 1740 (1992); J. J. Regan, S. M. Risser, D. N. Beratan, and J. N. Onuchic, J. Phys. Chem., 97 13083 (1993)), but do not travel through vacant space (S. M. Risser, D. N. Beratan, and T. J. Meade, J. Am. Chem. Soc., 115 2508 (1993)). This finding was later modified to include electron transfer between .pi.-stacked electron systems (F. Barigelletti, L. Flamnigni, V. Balzani, J. P. Collin, J. P. Sauvage, A. Sour, E. C. Constable, and A. C. M. W. Thompson, J. Am. Chem. Soc., 116 7692 (1994); J. N. Onuchic and D. N. Beratan, J. Am. Chem. Soc., 109 6771 (1987)). Subsequently, several groups measured rates of electron transfer through electroactive proteins (enzymes) using modified or unmodified electrodes, then microelectrodes (Hill, H. A. O., Klein, N. P., Murthy, A. S. N., Psalti, I. S. N., Chemical Sensors and Instrumentation, (1989) pp. 105-113; Armstrong, F. A., Bond, A. M., Hill, H. A. O., Psalti, I. S. N., Zoski, C. G., J. Phys. Chem. 93, (1989) pp. 6485-6493).
One drawback in these studies is that direct adsorption of protein onto an electrode typically resulted in loss of conductivity, presumably due to protein denaturation. A hydrophilic molecule (promoter) therefore was adsorbed to an electrode prior to adsorption of the electroactive protein in some cases. The promoter layer is designed to bind the protein of interest through hydrogen bonds, giving the electrons a suitable pathway. Notably, in these studies, electrons were observed to travel through an inert molecule, then through the electroactive molecule (Hill, H. A. O. and Lawrence, G. A., "Some Consequences of Mixed and Dilute Surface Modification of Gold Electrodes for Protein Electrochemistry," J. Electroanal. Chem. 270 (1989) pp. 309-318). The amplitude of the signal was dependent upon the potential difference.
Rates of electron transfer have also been measured through DNA (C. J. Murphy, M. R. Arkin, Y. Jenkins, N. D. Ghatlia, S. H. Bossman, N. J. Turro and J. K. Barton, Science, 262 1025 (1993)). It has been shown that the rate of electron transfer through double-stranded DNA is much faster than through single-stranded DNA (T. J. Meade and Jon F. Kayyem, Angew. Chem. Int. Ed. Engl., 34 3, pp. 352-354 (1995), "Electron Transfer through DNA: Site-Specific Modification of Duplex DNA with Ruthenium Donors and Acceptors").
Co-pending, commonly-owned U.S. patent application Ser. No. 08/312,388, filed Sep. 26, 1994 by Bamdad, et al., describes a technique for immobilization of single-stranded DNA at a surface as part of a self-assembled monolayer, and use of the arrangement in determination of biological binding partners of the DNA via Surface Plasmon Resonance (SPR), a technique that measures the very slight changes in mass that occurs at a surface upon biological binding of a binding partner to the surface-immobilized species.
Accordingly, it is an object of the invention to provide techniques for studying molecular interactions at surfaces.